The goal of this research is to extend our knowledge of gene organization and expression of tRNA in Escherichia coli. Techniques of two dimensional gel electrophoresis, in situ hybridization, and autoradiography will be employed to examine the arrangement of total tRNA species and specific leucyl tRNA in chromosomal DNA, with respect to specific endonuclease restriction sites, ribosomal RNA, and Insertion sequences. Using this approach, the in vivo expression of total, then specific leucyl tRNA will be assessed as a function of the physiological state of the cell and in the background of specific mutations (SuA, relA) which may be involved in tRNA regulation. Individual leucyl tRNA genes will be partially purified using RPC-5 chromatography, and preparative gel electrophoresis then cloned using lamba and/or plasmid vectors. A major thrust of this research will be the elucidation of the primary sequence of leucyl tRNA cistron(s) and particular emphasis will be placed on tRNAleu. Ultimately templates bearing these genes will be used as tools for studying detailed mechanisms of regulation using both in vivo and in vitro approaches. An additional goal of this proposed research will be to develop in vitro systems for the synthesis and detection of specific leucyl tRNA's to include a purified transcription system, tRNA dependent in vitro DNA directed transcription translation systems, and DNA templates containing a functional tRNA operator promoter fused to the beta galactosidase gene. Ultimately these systems will be used to investigate detailed mechanisms of regulation.